Effects of alpha-ketoglutarate on neutrophil intracellular amino and alpha-keto acid profiles and ROS production.

The aim of this study was to determine the effects of alpha-ketoglutarate on neutrophil (PMN), free alpha-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O(2) (-)), hydrogen peroxide (H(2)O(2))] and released myeloperoxidase (MPO) activity. Exogenous alpha-ketoglutarate significantly increased PMN alpha-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular alpha-ketoglutarate changes, increases in O(2) (-) formation, H(2)O(2)-generation and MPO activity have also been observed. We therefore believe that alpha-ketoglutarate is important for affecting PMN "susceptible free amino- and alpha-keto acid pools" although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic alpha-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.

Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and alpha-keto acid homeostasis?

We examined the effects of beta-alanine (taurine analogue and taurine transport antagonist), taurine (regarding its role in neutrophil (PMN) immunonutrition) and taurine combined either with L-NAME (inhibitor of *NO-synthase), SNAP (*NO donor), DON (glutamine-analogue and inhibitor of glutamine-requiring enzymes), DFMO (inhibitor of ornithine-decarboxylase) and beta-alanine on neutrophil amino- and alpha-keto acid profiles or important PMN immune functions in order to establish whether taurine transport-, nitric oxide-, glutamine- or ornithine-dependent mechanisms are involved in any of the taurine-induced effects. According to the present findings, the taurine-mediated effect appears to be based primarily on a modulation of important transmembraneous transport mechanisms and only secondarily on directly or indirectly induced modifications in intragranulocytic amino- and alpha-keto acid homoeostasis or metabolism. Although a direct relation to the parallel observed immunological modifications can only be presumed, these results show very clearly that compositional modifications in the free intragranulocytic amino- and alpha keto-acid pools coinciding with changes in intragranulocytic taurine levels are relevant metabolic determinants that can significantly influence the magnitude and quality of the granulocytic immune response.

Morphine inhibits AP-1 activity and CD14 expression in leukocytes by a nitric oxide and opioid receptor-dependent mechanism.

BACKGROUND: Activator protein 1 is a transcription factor involved in the regulation of proinflammatory mediators. Activation of phagocytes by lipopolysaccharide depends on the expression of CD14 on the cell surface. In this study, we investigated the effects of morphine and nitric oxide on CD14 expression and activator protein 1 activation in human blood monocytes and neutrophils as well as the leukocyte cell line HL-60. METHODS: Whole blood was incubated with morphine, the nitric oxide donor S-nitroso-N-acetyl-penicillamine, naloxone or nitric oxide synthase inhibitors Nomega-nitro-l-arginine and Nomega-nitro-l-arginine-methylester and stimulated with lipopolysaccharide. Activator protein 1 nuclear content was determined by flow cytometry in human blood neutrophils and monocytes. CD14 expression on neutrophils was measured after incubation with fluorescein isothiocyanate-labelled antibodies. Electric mobility shift assay served for evaluation of activator protein 1 nuclear binding in HL-60 cells. RESULTS: Incubation of whole blood with morphine and subsequent stimulation with lipopolysaccharide decreased activator protein 1 nuclear content. Exposure to naloxone before morphine treatment abolished morphine-induced inhibition of activator protein 1 activity in human blood monocytes and neutrophils. Nitric oxide synthase inhibitors also reversed morphine's effects. CD14 expression on neutrophils was reduced after morphine treatment. These effects were antagonized by nitric oxide synthase inhibitors and naloxone. CONCLUSION: Morphine inhibits activator protein 1 activation by a mu opioid receptor pathway coupled to nitric oxide as second messenger. The decrease in CD14 expression caused by morphine may play a role in inhibition of activator protein 1 activation following lipopolysaccharide treatment of phagocytes.

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and alpha-keto acid homeostasis or immunocompetence.

We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, beta-alanine and DFMO on neutrophil amino and alpha-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of *NO-synthase [L-NAME], an *NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [beta-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.

Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and alpha-keto acid profiles or host defense capability.

We have examined the effects of N(omega)-nitro-L-arginine-methylester-hydrochloride [L-NAME; inhibitor of nitric oxide synthase], S-nitroso-N-acetyl-penicillamine [SNAP; nitric oxide donor], alpha-difluoro-methyl-ornithine [DFMO; inhibitor of ornithine decarboxylase] arginine or ornithine as well as the combination of arginine or ornithine with L-NAME, SNAP or DFMO on intracellular free amino- and alpha-keto acid profiles and the immune function markers superoxide anion and hydrogen peroxide generation as well as released myeloperoxidase activity in neutrophils (PMN). Although the underlying mechanisms still remain unclear, we believe from our results that nitric oxide as well as polyamine-dependent pathways are involved in the signal transmission of free radical molecule, beneficial nutritional therapy or maleficient pharmacological stress-induced alterations in PMN nutrient composition. Relevant changes in intragranulocyte free amino- and alpha-keto acid homeostasis and metabolism, especially, may be one of the determinants in PMN nutrition that positively or negatively influences and modulate neutrophil host defence capability and immunocompetence.

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine.

The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free alpha-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN alpha-ketoglutarate, pyruvate PMN superoxide anion (O2-) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in alpha-ketoglutarate, pyruvate, MPO release and H2O2 generation. Formation of O2- on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and alpha-ketobutyrate levels, decreased O2- and H2O2 formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-alpha-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.

[Small pox--infection, therapy and anaesthesiological management (part 1)]. / Pocken--Klinik, Therapie und anästhesiologische Aspekte (Teil 1).

Smallpox is an acute contagious and sometimes fatal infectious disease. It is caused by the variola-virus. Smallpox is characterized by a typical disease form with a progressive distinctive skin rash, especially at face, arms and legs. Smallpox has a fatality rate of about 30 % and the therapy of infected patients is only symptomatically. As prevention the WHO initiated worldwide vaccination programs in the year 1967. The last naturally occurring case of smallpox in the world was in Somalia in 1977. Since then the only known cases of smallpox happened from an outbreak in Birmingham, England caused by a laboratory accident in the year of 1979. On May the 8 th 1980 the disease was declared as eliminated from the world by the WHO (WHO-Resolution 33.33). A natural occurrence of the variola-virus seems to be not given. Nevertheless the virus exists for research in two laboratories, the American Centers of Disease Control and Prevention in Atlanta, Georgia and in the Russian Research Center for Virology and Biotechnics in Kolzowo, Sibiria. Threatening infections with smallpox or other microorganisms, used as bioweapons, get a new dimension through global terrorism. The variola-virus represents an optimal candidate for bioweapons. It is easy to replicate, it is highly contagious and the transmission over aerosol or direct contact from man to man is easy to handle. After the disease was eliminated from the world, routine vaccination among general public was stopped. Therefore younger people don't possess any vaccination protection. Older formerly vaccinated people probably have only a non-sufficient protection. Because of the smallpox elimination a lot of physicians have no experience with this disease. An outbreak of this smallpox isn't only controlled by new vaccination. In our times we need adapted prevention-standards, pox-alarm plans and quarantine standards.

Inhibitory effects of S-(-) and R-(+) bupivacaine on neutrophil function.

BACKGROUND: Local anesthetics inhibit migration, enzyme release and superoxide anion generation of polymorphonuclear leukocytes (PMN). Due to their ability to phagocytose and kill bacteria PMN represent a major defense mechanism in the circulating blood. In this study we determined the influence of racemic bupivacaine and its enantiomers on neutrophil phagocytic activity, oxidative burst as well as surface expression of complement and Fcgamma receptors. METHODS: Venous blood was pre-incubated with different concentrations of either racemic bupivacaine, R-(+) or S-(-) bupivacaine. Fluoresceine isothiocyanate (FITC)-labeled antibodies against Fcgamma receptor III (CD16), complement receptor 1 (CD35) and complement receptor 3 (CD11b) were used to determine surface receptor expression. Phagocytic activity was measured by ingestion of FITC-labeled vital Staphylococcus aureus. Oxidative burst was determined by conversion of nonfluorescent dihydrorhodamine 123 into fluorescent rhodamine 123. Fluorescent intensity of each sample was determined by flow cytometry. RESULTS: Racemic bupivacaine inhibited surface receptor expression, phagocytosis, and oxidative burst in a time- and concentration-dependent manner. Although the S-(-) enantiomer exerted significantly less inhibitory action on neutrophil function compared to R-(+) and racemic bupivacaine, these effects were small compared to the overall changes. CONCLUSION: These findings suggest that bupivacaine impairs surface receptor expression and may thereby contribute to reduced phagocytic activity and oxidative burst. Enantiomer-specific effects of bupivacaine may play a minor role in the inhibition of these leukocyte functions.

[Transmissible spongiform encephalopathies--anesthesiologic and intensive care management]. / Ubertragbare spongiforme Enzephalopathien--Anaesthesiologisches und intensivmedizinisches Management.

The transmissible spongiform encephalopathies (TSE) are known to affect humans and various animals. The bovine spongiform encephalopathy (BSE) and the human Creutzfeldt-Jacob disease (CJD) are among the most notable degenerative disorders caused by prions. Considering the BSE epidemic and the description of a new variant of Creutzfeldt-Jacob disease (nvCJD), which is probably related to bovine spongiform encephalopathy, TSE have recently gained a lot of public attention. Although the causative factors (prions, viruses) are still under discussion, none of the present concepts are explanatory for all aspects of the human CJD. CJD may present as a sporadic, genetic, or infectious illness and there is now considerable concern that bovine prions may have been passed to humans. To exclude transmission of CJD via medical products and instruments, the effectiveness of cleaning, disinfection and sterilization procedures must be firmly established. This manuscript presents an overview to anaesthesiology and intensive care medicine of recommended inactivation procedures and assessed these procedures in the light of the inactivation of prions.

Morphine suppresses complement receptor expression, phagocytosis, and respiratory burst in neutrophils by a nitric oxide and mu(3) opiate receptor-dependent mechanism.

We investigated whether morphine and fentanyl influence surface receptor expression, phagocytic activity and superoxide anion generation of neutrophils in a whole blood flow cytometric assay. Morphine suppressed complement and Fcgamma receptor expression and neutrophil function in a concentration- and time-dependent manner. Morphine-induced changes were similar to those caused by the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine and were abolished by preincubation with the NO synthase inhibitor N-nitro-L-arginine as well as naloxone. Fentanyl had no immunosuppressive effects. These results suggest that these neutrophil functions are inhibited by morphine-stimulated NO release mediated by the mu(3) opiate receptor subtype found on immunocytes.

[The effect of different volume expanders on neutrophil granulocyte function in vitro]. / Der Einfluss verschiedener Volumenersatzmittel auf die Funktion von neutrophilen Granulozyten in vitro.

INTRODUCTION: The influence of kolloids on the immune system is not well documented. In this study we investigated the effects of gelatine, hydroxyethylstarch (HES), human albumine, and dextrane on neutrophil function and receptor expression by flow cytometry. METHODS: Whole blood of healthy volunteers was incubated for 30 minutes with either gelatine, HES (6% and 10%), dextrane 40 and 60, or human albumin 20%. Phagocytic capacity was determined by uptake of fluorescein-isothiocyanate labeled bacteria, the conversion of dihydrorhodamine 123 into fluorescent rhodamine 123 was used for oxidative burst measurements. Expression of complement receptors CD 11b and CD35 was investigated using fluorescein-isothiocyanate labeled antibodies. RESULTS: Incubation with gelatine significantly increased expression of complement receptors and oxidative burst. Dextranes and HES had no influence on neutrophil function. Human albumin reduced the oxidative burst, whereas CD 35 expression was increased. CONCLUSION: The physiological significance of these changes in a range of 10% has to be clarified in further investigations.

[Susceptibility to infections in diabetes--effects on metabolism]. / Infektanfälligkeit bei Diabetes--Einfliüsse auf den Stoffwechsel.

Diabetic patients suffer from recurrent episodes of infections. The cellular and the humoral elements of the defense system against germ invasion are disturbed by the diabetic metabolism. Neuropathy and vascular damage promote the development of wounds and inhibit their healing. Altered motility of the gastrointestinal and the urinary tract lead to increased penetration of bacteria even there. Rare bacteria, atypical courses and frequent complications of infections result in delayed diagnosis and therapy. Dehydration, electrolyte disturbances, malnutrition, and reduced general conditions even increase susceptibility to an infection. On the other hand, an infection deteriorates the metabolic situation in diabetes, resulting in the need for higher insulin doses, or insulin injections in patients normally on oral medication. Altered every-day-life with modified food intake and reduced physical activity complicate diabetes therapy. Neuropathy, angiopathy, retinopathy, nephropathy and other diabetic complications can be triggered and aggravated during the course of an infection. To disrupt this vitious circle of hyperglycemia enforcing infections, which then raise blood glucose, it is necessary to know about the characteristic features of the interactions of diabetes and infection.